Hypoxia Promoting Induction Of Regulatory T Cells In Gastric Cancer And Intervention Mechanism Of Wogonin

The regulatory T cell (Treg) constitute approximately5-10%of peripheral CD4+T cells in healthy individuals, and have recently received widespread attention from immunologists. Increased Tregs have been reported in the circulation and tumor tissue of patients with various cancers including gastric cancer. Accumulating data also indicated that the presence of Treg induce an increased risk for progression of cancer. Tregs can inhibit the function of immune cells including CD4+T, CD8+T, NK and NKT to promote immune tolerance.Tregs-mediated immunosuppression is considered to be one of the crucial tumor immune-evasion mechanisms and the main obstacle for tumor immunotherapy. Intratumor hypoxia is a common feature of solid tumors including gastric cancer, which might influence the progression of tumor by activating key biochemical and cellular pathways. In recent years, studies have found wogonin can block hypoxia-associated molecular pathways and regulate immune cell activity, thus exert anti-tumor activity.In this study, we hypothesized that gastric cancer may acquire a selective advantage by induction of Tregs under hypoxia, thereby evading immune surveillance. To demonstrate our hypothesis, we analyzed the expression of hypoxia inducible factor-la (HIF-la) and Foxp3in gastric cancer tissues, detected the changes in TGF-β1and Treg cells before and after surgery in the peripheral blood of gastric cancer patients, assessed the effect of hypoxia on TGF-β1production in cancer cell lines, and finally elucidated the role of hypoxia mediating Tregs enrichment in tumor. In addition, it will be verified whether wogonin inhibits the ability of inducing Tregs in preliminary ex vivo examination.This study will elaborate the relationship between tumor hypoxia and tumor immune escape, and have important theoretical implications for anti-tumor immunotherapy looking for new molecules. In addition, mechanisms of anti-tumor activity of wogonin would be elucidated deeply.Our study was divided into four parts.Part Ⅰ. HIF-1α and Foxp3expression in gastric cancer tissuesObjective:To analyze the relationship between HIF-1α and Foxp3in gastric cancer tissue, to detect the association between HIF-1α or Foxp3and clinicopathologic characteristics, and to clarify the relationship between OS and HIF-1α or Foxp3. Methods:We investigated the expression of HIF-la and Foxp3by immunohistochemistry in99patients. HIF-1α was evaluated using staining score. The mean number of Foxp3+Treg cells was calculated. We then performed immunohistochemical double-staining to evaluate the presence of coexpression between HIF-1α and Foxp3or TGF-β1.Results:HIF-la was found predominantly in the cytoplasm and nucleus of cancer cells at the tumor margin and only nuclear HIF-1α staining was scored.In some cases, lymphocytes also showed strong expression for HIF-la. Most of Foxp3positive cells were also located at the tumor margin where close to the cells expressing HIF-la. The number of Foxp3+cells and score of HIF-la in gastric carcinoma was significantly higher than that in adjacent tissues. A strong positive correlation between the number of Foxp3+cells and HIF-la expression was observed in tumor tissue. There was an increase in the positive rate of HIF-la with depth of tumor invasion. A similar trend was also found between the expression of HIF-la or Foxp3and tumor stage. Additionally, a significant correlation was found between lymph node metastasis and the expression of HIF-1α or Foxp3. In our study, OS in Foxp3-low and HIF-la-negative group was significantly higher than in Foxp3-high and HIF-la-positive group. The results of immunohistochemical double-staining showed that Foxp3+cells mainly assemble in the sites close to hypoxic tumor region with high HIF-la expression. Furthermore, most high HIF-la tumor cells were high expression of TGF-β1.Conclusions:Intensity of HIF-la expression was positively correlated with density of Treg infiltration in gastric cancer tissue. The expression of HIF-la and Foxp3in tumor tissues was positively with unfavorable clinicopathological features and poor prognosis. Foxp3+cells mainly assemble in the sites close to hypoxic tumor region. Intratumor hypoxia and TGF-β1may promote Treg infiltration in gastric cancer tissue.Part Ⅱ. Circulating Tregs and the concentration of serum TGF-β1in pre-surgery and post-surgery patientsObjective:To explore effects of tumor on the circulating Tregs and the concentration of TGF-p1in peripheral blood of patients with gastric cancer. To clarify the relationship between the concentration of TGF-β1and the circulating Tregs. To provide evidence for the role of TGF-β1proved a bridge connecting tumor hypoxia and Tregs.Methods:Peripheral bloods were collected from20patients with gastric cancer one day before and10days after surgery from July2012to October2012. CD4+Foxp3+T cells were analyzed in patients with gastric cancer via flow cytometry. Serum TGF-β1concentration was detected via ELISA. Results:A significant correlation was found between lymph node metastasis and the proportion of Tregs in peripheral bloods. A similar trend was also found between the concentration of TGF-β1and poorly differentiated type pathology. A positive correlation was found between Tregs frequency and TGF-β1concentration in serum before operation. The frequency of Tregs before surgery was higher than that after surgery. TGF-β1concentration after surgery decreased significantly when compared with those before surgery.Conclusions:There was a positive correlation between Tregs frequency and TGF-β1concentration in peripheral blood of patients with gastric cancer. A correlation was found between poorly clinical and pathological features correlation and Tregs frequency or TGF-β1concentration in peripheral blood. Surgical removal of the tumor may reduce the proportion of Tregs and the concentration of TGF-β1in peripheral blood of patients with gastric cancer. These results suggested that the tumor affects the body’s immune characteristics.Part Ⅲ. Hypoxia augments the ability of inducing CD4+Foxp3+T cells via TGF-β1in gastric cancer cell linesObjective:To observe effects of supernatants from gastric cancer cells under different condition on chemotaxis and expansion for Tregs ex vivo, to detect effects of hypoxia on the expression of TGF-β1in gastric cancer cell lines and to verify that hypoxia augments the ability of inducing CD4+Foxp3+T cells via TGF-β1.Methods:Human gastric cancer cell lines, AGS and BGC823, were propagated in5%CO2at37℃for24h under hypoxic (1.0%O2) or normoxic (21%O2) conditions. The supernatants collected from cultured AGS and BGC823cells under normoxic or hypoxic condition diluted with fresh AIMV medium (1:3) called normoxic medium and hypoxic medium respectively. The chemotaxis of normoxic medium and hypoxic medium on Treg cells in PBMC was observed in Transwell experiments. The expansion of normoxic medium and hypoxic medium on Treg cells in PBMC was observed in coculture systems. The expression of TGF-β1was also detected by qPCR, ELISA and immunofluorescence in two human gastric cancer cell lines under hypoxic or normoxic conditions. TGF-β receptor I phosphorylase inhibitor, recombined TGF-β1and other condition mediums were used to culture isolated CD4+CD25T cells. Flow cytometry were used to detect the Foxp3+Tregs.Results:The results in chemotaxis experiments show that the Foxp3expression of T cells in normoxic medium medium was increased as compared with control medium,while the Foxp3expression in hypoxic medium was not different from that in normoxic medium. The results in coculture systems show that the Foxp3expression of T cells in normoxic medium and hypoxic medium was increased as compared with control medium, while the Foxp3expression in hypoxic medium was significantly higher than in normoxic medium. The mRNA expression of TGF-β1under hypoxia was increased as compared with under normoxia. Consistent with qPCR, the TGF-β1level in the culture supernatant was also shown same results. Further, the expression of TGF-β1was also detected by immunofluorescence, and the result showed increased TGF-β1expression under hypoxic condition when compared with under normoxia.In induction experiments, the Foxp3expression of T cells in normoxic medium, hypoxic medium and recombinant human TGF-β1medium was increased as compared with control medium. In addition, Foxp3expression in hypoxic medium was significantly higher than in normoxic medium. Further, as LY-364947was added to each kind of medium, the Foxp3expression decreased overtly.Conclusions:The supernatants of gastric cancer cells could promote the ability of chemotaxis and expansion for Tregs. Hypoxia promotes the ability of expansion,but has no effect on ability of chemotaxis. Hypoxia promotes immune tolerance by inducing regulatory T cells via TGF-β1in gastric cancer immune tolerance by inducing regulatory T cells.Part IV Effects of wogonin on TGF-β1 secretion of gastric cancer cells and on induction of TregsObjective:To observe effects of wogonin on TGF-β1 secretion of gastric cancer cells ex vivo and to verify whether wogonin inhibits the ability of inducing CD4+Foxp3+T cells via TGF-β1.Methods:Human gastric cancer cell lines, AGS and BGC823, were propagated in5%CO2at37℃for24h under hypoxic (1.0%O2) or normoxic (21%O2) conditions. Wogoins were added into AGS and BGC823cell culture system for24hours. TGF-β1concentration in culture supernatants were detected by ELISA. The supernatants collected from cultured AGS and BGC823cells were made into condition medium to culture culture isolated CD4+CD25"T cells. Flow cytometry were used to detect the Foxp3+Tregs.Results:Under normoxic conditions, Wogonin had on effect on TGF-β1 secretion of AGS cells, but inhibited TGF-β1secretion of BGC823cells. Under hypoxic conditions,wogonin inhibited TGF-β1secretion both of BGC823cells and AGS cells. Cultured in normoxic condition meidiums, there were no difference of CD4+Foxp3+T cells proportion between wogonin and solvent groups. Cultured in hypoxic condition meidiums, the proportion of CD4+Foxp3+T cells in wogonin groups was lower than that in solvent groups.Conclusions:Wogonin not only suppressed TGF-β1expression caused by hypoxia in gastric cancer cells, but also inhabited the induction of Tregs.In conclusion, the present study demonstrated that hypoxia potentiates the ability of gastric cancer to evade immune surveillance by up-regulating expression of TGF-β1, thereby induction of Tregs in tumor. Further, our data also provided the evidence for the existence of intercellular cross-talk between the tumor cells and Tregs, which might regulate anti-tumor immune responses. Preliminary results confirmed that wogonin not only suppressed TGF-β1expression caused by hypoxia in gastric cancer cells, but also inhabited the induction of Tregs.